| Objective:WHO reported, the infertility incidence of couples of childbearing age in the developed countries was 15% and male infertility accounted for 50%. Male infertility could be caused by systemic endocrine diseases, sperm production obstacles and transportation obstacles in reproductive duct, and the main reason was sperm production obstacles among them.The objectives of the further study were to investigate the molecular mechanism of sperm production obstacle after heating shock, to confirm the role of heat shock proteins in the process of germ cell apoptosis, and to clarify the molecular targets of wuzi yanzong pill on sperm production obstacle so that to broaden the clinical application range of traditional Chinese medicine in male infertility caused by sperm production obstacle.Methods:Experiment one:66 male rats were randomly divided into the normal group,10 min heating shock group and 20 min heating shock group. And the testes of rats were immersed in a waterbath at 43℃(or 22℃) for 10 min or 20 min. Rats in heating shock groups were anaesthetized and testes were extracted for further analysis after heating shock 0.5 h, 2 h,6 h,24 h,48 h respectively. Morphological changes were observed by the paraffin section of HE staining. Real time PCR was used for determining the expressions of Hsp70, Hsp90, Hsp105, Bax, Bcl-2 mRNA.Experiment two:36 male rats were randomly divided into the normal group and the heat shock group. And the testes of rats were immersed in a waterbath at 43℃(or 22 ℃) for 20 min. Rats in the heat shock group were anaesthetized and testes were extracted for further analysis after heating shock 0.5 h,2 h,6 h,24 h,48 h respectively. Morphological changes were observed by the paraffin section of HE staining and TdT-mediated dUTP nick end labeling (TUNEL) technique. Real time PCR was used for determining the expressions of Hsp70, Hsp90, Hspl05, Bax, Bcl-2 mRNA. Western blot was used to detect the expression of cleaved caspase-3.Experiment three:102 male rats were randomly divided into five groups, i.e., normal group, model group, low-dose Wuzi Yanzong pill group, medium-dose Wuzi Yanzong pill group and high-dose Wuzi Yanzong pill group. And the testes of rats were immersed in a waterbath at 43℃ (or 22℃) for 20 min. On days 0,5,10 and 15, six rats respectively in all groups were anaesthetized and their testes were extracted for further analysis. Hormone assays were measured by RIA. Morphological changes were observed by the paraffin section of HE staining. Real time PCR was used for determining the expressions of Hsp70, Hsp90, Hsp105, Bax, Bc1-2 mRNA.Results:Experiment one:There were no obvious changes of seminiferous epithelium in all groups after heating shock (43 ℃,10 min). By contrast, extensive changes were observed in those heated to 43 ℃,20 min with multinucleated giant cells and degenerating germ cells observed within the seminiferous epithelium at 24 h. At 48 h, germ cell depletion was evident and large areas of the seminiferous epithelium only contained Sertoli cells, spermatogonia and elongated spermatids. At 0.5 h, Hsp70 mRNA expression after 20 min heating shock was about three times as much as the model group 0.5 h after 10 min heating shock, and 2 h reached 3.5 times.6 h after heating shock, Hsp70 mRNA level of group 10 min had restored to normal level, but 20 min heat shock model group still remained high expression. Similarly, the expressions of Hsp90, Hsp105, Bax, Bc1-2mRNA, Bc1-2/Bax in groups 20 min changed greater than those in groups 10min.Experiment two:We found that the loss of germ cells occurred gradually and the apoptosis signal also increased significantly after heating shock. There were no obvious changes of seminiferous epithelium at 0.5 h and 2 h after heating shock while positive cells in group 6 h was significantly increased. Apoptosis occurred 24 h at the worst, but the number of positive cells in group 48 h decreased, which were consistent with morphological changes. The expression of Hsp70 mRNA quickly increased after heating shock, and the differences of Hsp70 mRNA expression between group 0.5 h or group 2 h and normal group were statistically significant (P<0.05). The expression of Hsp90 mRNA decreased after heating shock, and the differences of Hsp90 mRNA expression between heat shock groups and normal group were statistically significant (P<0.05). While the expression of Hsp105 mRNA increased after heating shock, and reached the highest at 6 h and had statistical significance (P<0.05). Compared with normal group, the differences of Bax mRNA expression in others groups were not statistically significant (P>0.05). However, the differences of Bcl-2 mRNA expression between group 0.5 h or group 2 h or group 6 h and normal group were statistically significant (P<0.05). More interestingly, the trend of Bcl-2/Bax expression also gradually rised after heating shock and the differences of Bcl-2/Bax expression between group 0.5 h or group 2 h or group 6 h and normal group were statistically significant (P<0.05). The minimum expression of actived caspase-3 was at 2 hand the difference was statistically significant compared with normal group.Experiment three:All the differences of plasma levels of FSH, LH, T among groups had no statistical significance (P>0.05). On histopathological examination, testes of control rat showed normal morphology and spermato gene sis, containing abundant amounts ofspermatids and sperms in the lumen. In contrast, after scrotal hyperthermia, the cells of model group were disturbed in the seminiferous tubules. Germinal epithelial cells were separated from each other and from the tubular basement membrane. In 5 days after scrotal hyperthermia, there were multinucleated giant cells and consequent appearance of irregular spaces in the epithelium. Germ cell loss was also obvious in testes at 10 day and many tubules appeared to lack both spermatocytes and round spermatids causing the seminiferous epithelium to collapse. In 15 days after scrotal hyperthermia, severe loss of germ cells and consequent appearance of small to large vacuoles in the epithelium were observed during this time, and they were surrounded by a thickened basal membrane. All the differences of Hsp70 mRNA expression among groups had no statistical significance (P>0.05). In 5 days after scrotal hyperthermia, the expression of Hsp90 mRNA decreased and the differences of Hsp90 mRNA expression between heat shock groups and normal group were statistically significant (P<0.05); and the differences of Hsp90 mRNA expression between the model groups of 5 days,10 days and the model group after heating shock were statistically significant (P<0.05); There are no significant differences among other groups (P>0.05). In 5 days after scrotal hyperthermia, the differences of Hspl05 mRNA expression between model group, low-dose group, high-dose group and normal group were statistically significant (P<0.05); There are no significant differences among other groups (P>0.05). In 5 days after scrotal hyperthermia, the differences of Bax mRNA expression between middle-dose group and normal group were statistically significant (P<0.05), and the differences of Bax mRNA expression between the model groups of 10 days,15 days and the model group after heating shock were statistically significant (P<0.05); There are no significant differences among other groups (P>0.05). In 5 days after scrotal hyperthermia, the differences of Bcl-2 mRNA expression between model group and normal group were statistically significant (P<0.05), and the differences of Bcl-2 mRNA expression between the model groups of 10 days,15 days and the model group after heating shock were statistically significant (P<0.05); There are no significant differences among other groups (P>0.05). The differences of Bc1-2/Bax expression between the model groups of 10 days,15 days and the model group after heating shock were statistically significant (P<0.05); There are no significant differences among other groups (P>0.05).Conclusions:Experiment one:A model of sperm production obstacles could be successfully established with rats after testicular heating shock at 43 ℃ with 20 min.Experiment two:Hsp70 mRNA, Hspl05 mRNA excessive expressions and Hsp90 mRNA downregulating expression were induced by testicular heating shock at 43 ℃ with 20 min, and they may play the antiapoptotic role through up-regulating the expression of Bcl-2/Bax and down-regulating the expression of actived caspase-3.Experiment three:The expressions of Hsp70, Hsp90, Hsp105 could not be reversed in the testis even though rats were given by gavage once a day for 5 days, lOdays or 15days with Wuzi Yanzong pill after testicular heating shock at 43 ℃ with 20 min. And Wuzi Yanzong pill could not inhibit germ cells from apoptosis in this experiment.
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